Geri Kreitzer

The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell–cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP–actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell–cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilinS3A inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA–GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells.

Epithelial polarization is associated with selective stabilization and reorganization of microtubule (MT) arrays. However, upstream events and downstream consequences of MT stabilization during epithelial morphogenesis are still unclear. We show that the anterograde kinesin KIF17 localizes to MT plus ends, stabilizes MTs, and affects epithelial architecture. Targeting of KIF17 to plus ends of growing MTs requires kinesin motor activity and interaction with EB1. In turn, KIF17 participates in localizing adenomatous polyposis coli (APC) to the plus ends of a subset of MTs. We found that KIF17 affects MT dynamics, polymerization rates, and MT plus end stabilization to generate posttranslationally acetylated MTs. Depletion of KIF17 from cells growing in three-dimensional matrices results in aberrant epithelial cysts that fail to generate a single central lumen and to polarize apical markers. These findings implicate KIF17 in MT stabilization events that contribute to epithelial polarization and morphogenesis.

A key process during epithelial polarization involves establishment of polarized transport routes from the Golgi to distinct apical and basolateral membrane domains. To do this, the machinery involved in selective trafficking must be regulated during differentiation. Our previous studies showed that KIF5B selectively transports vesicles containing p75-neurotrophin receptors to the apical membrane of polarized, but not non-polarized MDCK cells. To identify the kinesin(s) responsible for p75 trafficking in non-polarized MDCK cells we expressed KIF-specific dominant-negative constructs and assayed for changes in post-Golgi transport of p75 by time-lapse fluorescence microscopy. Overexpression of the tail domains of kinesin-3 family members that contain a C-terminal pleckstrin homology (PH) domain, KIF1A or KIF1Bβ, attenuated the rate of p75 exit from the Golgi in non-polarized MDCK cells but not in polarized cells. Analysis of p75 post-Golgi transport in cells expressing KIF1A or KIF1Bβ with their PH domains deleted revealed that vesicle transport by these motors depends on the PH domains. Furthermore, purified KIF1A and KIF1Bβ tails interact with p75 vesicles and these interactions require the PH domain. Knockdown of canine KIF1A also inhibited exit of p75 from the Golgi, and this was rescued by expression of human KIF1A. Together these data demonstrate that post-Golgi transport of p75 in non-polarized epithelial cells is mediated by kinesin-3 family motors in a PH-domain-dependent process.